Fluorescent protein development 1) We are studying photoswitching behaviors of photoswitchable fluorescent proteins and their use in Forster Resonance Energy Transfer (FRET) experiments. This includes an ongoing project to develop photoswitchable yellow fluorescent proteins, better photoswitchable red fluorescent proteins, and far-red fluorescent proteins for use in conventional diffraction-limited microscopy as well as super-resolution molecular localization microscopy. Current emphases are on developing multi-label FRET experiments(3-4 interacting proteins) using combinations of psFRET and acceptor photoswitching methods and well as developing mutants with disparate photoswitching kinetics. 2) We continue our collaboration with Joy Zhao and Peter Schuck on surveying numerous fluorescent proteins to better define their oligomerization characteristics. These characteristics have not been rigorously determined and unforeseen oligomerization leads to aberrant fluorescent protein behavior. Our efforts are to develop variants with little to no self-association behaviors. 3) We have an ongoing project to develop improved red fluorescent proteins. Current variants display low fluorescence, slow maturation, and/or oligomerization. Biochemical analyses of wild type proteins coupled with site-directed mutagenesis has led to our discoveries of mRuby and Scarlet variants with much decreased self-association, increased brightness, and faster maturation. We have upgraded our instrumentation and data analyses to better screen mutant forms expressed in bacteria colonies growing on agar plates. Cell biology projects 1) Gaetan Herbomel, a former post-doctoral fellow in the lab of Lawrence Tabak, works in my lab and is completing a project to image the localization of Golgi apparatus enzymes using Stochastic Optical Reconstruction Microscopy (STORM). The multi-color super-resolution experiments have required development an ImageJ plugin for single molecule co-localization analyses. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. We are currently developing electron microscopy methods in collaboration with Maria Aronova and Richard Leapman in our efforts to more precisely determine the relative localizations of these molecules. 2) We collaborate with Jim Kochenderfer (NCI) to study the self-association properties of chimeric antigen receptor (CAR) molecules using our psAFRET technique (see below). 3) We are utilizing our psFRET technique in conjunction with photoswitchable fluorescent protein tagged histone proteins to determine the binding sites of anti-cancer drugs, such as doxorubicin, within the chromatin of live cells. In related studies, we are also monitoring the proximity of PS-FP tagged histone proteins during chromatin compaction using psAFRET. Instrumentation and imaging development 1) Forster Resonance Energy Transfer (FRET) is a powerful approach to study the interactions of fluorescent molecules. We have developed new approaches which can be performed on a conventional widefield or confocal microscope to image FRET between a donor photoswitchable fluorescent protein, Dronpa, and an acceptor based on a conventional fluorescent protein. The technique which we call photoswitching FRET (psFRET) is based on photoswitching kinetics of a photoswitchable fluorescent protein in the presence and absence of an acceptor. 2) Another FRET based project using psFPs derived directly from the psFRET studies involves imaging homo-FRET by monitoring the anisotropy of the PS-FPs during the photoswitching process as a read-out of protein-protein interactions. This technique, which we called photoswitching anisotropy FRET (psAFRET), differs from measurements of hetero-FRET (energy transfer between two different color proteins) since it monitors energy transfer between copies of the same probe. 3) We have developed and continuously upgrade a series of image analysis macros that can be run within the ImageJ application to analyze psFRET and psAFRET data. To make these techniques more widely useful we have developed ImageJ plugins that are more versatile and can be used on almost any dataset. In addition, plugins available on the ImageJ update site (https://imagej.net/User:Pattersg) to analyze the exponential decay in fluorescence at each pixel as the photoswitchable fluorescent protein switches off. 4) We collaborated with the Hari Shroff lab on the development of improved forms of TIRF microscopy. These have resulted in improvements to lateral super-resolution levels while maintaining the acquisition speeds necessary for live cell imaging. Our collaboration is now focused on improving axial resolution through the use of photoswitchable fluorescent proteins.